The concept of cancer stem cells and their potential impact on chemotherapeutic responses is an area of intense interest in cancer research. One such agent, erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), is currently used for treatment of non-small cell lung cancer (NSCLC), however, the response is incomplete in even highly sensitive tumors and the basis for this is not known. To test the hypothesis that resistant sub-populations of cells of may be responsible for the incomplete response, the H1650 and H1975 NSCLC cell lines were sorted on the basis of CD24 and CD44 surface marker expression. In both cases, cell lacking both CD24 and CD44 expression (CD24-/CD44-), were the most resistant to erlotinib treatment in clonogenic assays. Neither the CD24-/CD44- phenotype nor relative resistance to erlotinib was associated with increased efflux of Hoechst 33342 dye, suggesting that this assay for putative stem cells could not explain drug sensitivity. However, CD24-/CD44- cells are characterized by differences in EGFR signaling pathways, supporting the hypothesis that non-small cell lung cancers may consist of heterogeneous populations of cells, with variable cell signaling activity and variable responses to erlotinib.We are currently awaiting the results from xenograft transplantation into NOD/SCID mice. Polychromatic immunophenotypic analysis has the capability to potentially resolve new clinically import lymphocyte subsets, while at the same time minimizing sample processing and the costs associated with the use of redundant antibodies in multiple tubes. Generally, the rule of thumb for selecting the appropriate fluorochrome combination is to assign the brightest label (PE, APC, or PE-Cy5) to the antibody that reacts with the protein of interest having the lowest expression. Multiple factors can affect the perception of brightness including the biophysical properties of the fluorochrome itself and instrument-dependent variables. Peridinin chlorophyll protein (PerCP), a subunit of the photosynthetic complex of the dinoflagellate Glenodinium sp., has an extremely high quantum efficiency and large Stokes shift, yet it photobleaches easily, thus limiting it usefulness in high power jet-in-air flow cytometers and restricting its use to low-power, fixed beam systems. PerCP is used for multicolor analysis since its spectral overlap is minimal with other common fluorochromes;however it commonly thought to be more suited for antigens expressed at medium or high densities. In the course of designing and testing a hybrid 4-color panel for routine immunophenotypic analysis of the major lymphocyte subsets (CD4 and CD8 T-cells, B, and NK cells), PerCP-conjugated antibodies could be readily utilized without any sacrifice in accuracy for common antigens expressed at relatively low densities, as in B-cells. However, staining was significantly altered when PerCP-conjugated antibodies to CD19 were used in stabilized blood samples. Additionally, cell fixation and permeabilization is a requirement for the measurement of intracellular antigens, as in the case of ZAP-70 expression, a surrogate marker in chronic lymphocytic leukemia disease progression and survival. Under these conditions, the accurate enumeration B-cells is preferentially and significantly altered, whether measured with PE-, PerCP, or PEAF700-conjugated fluorochromes. Overall, this diminished sensitivity leads to a poorer separation between the background fluorescence of unlabeled- (control) and labeled-cells, thereby confounding the accurate measurement of B-cell numbers and introducing error in the validity of the clinical value of these markers stained under these conditions. In addition to this study, we have examined the stability of B-cell markers using commercially available cell stabilization reagents to stabilize cell morphology and preserve cellular antigen expression.